Background
Triple-negative breast cancer (TNBC) is usually characterized by an aggressive phenotype, associated with an increased risk of early recurrence within 3 years after diagnosis, and poor prognosis [
1]. Current treatment approaches are limited to cytotoxic chemotherapy due to the lack of specific therapeutic targets [
1]. Therefore, the identification of reliable prognostic markers and novel therapeutic targets may allow a better stratification of patients with TNBC, and provide the rationale for investigating innovative treatment strategies.
Recent evidence indicates that the immune microenvironment plays a key role in cancer progression and response to therapies [
2]. Notably, the presence of tumor-infiltrating lymphocytes (TILs) is emerging as an important predictor of outcome and response to chemotherapy in TNBC [
3‐
7]. However, the composition of the tumor immune microenvironment is very heterogeneous, and the functional significance of specific immune cell subpopulations remains poorly understood. Indeed, cytotoxic CD8+ T cells have been shown to be an independent favorable prognostic factor, while studies on CD4+ T helper and forkhead box protein 3 (FOXP3) + T regulatory cells have shown conflicting results [
8].
Furthermore, even though TILs are able to identify and eliminate malignant cells, tumors have developed multiple mechanisms to maintain an immunosuppressive microenvironment, including the upregulation of inhibitory receptors, such as programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) [
9]. Recent findings demonstrate that the expression of immune markers related to immunosuppression is enriched in triple-negative/basal-like breast cancer, and correlate with prognosis and response to chemotherapy, supporting the evaluation of immunotherapy in TNBC [
6,
10‐
14]. Thus, a deeper understanding of the composition and the functionality of lymphocytic infiltration could be useful to predict patients’ outcome, and to select patients with TNBC who may benefit from the addition of immune checkpoint drugs to standard chemotherapeutic regimens. The aim of the present study was to evaluate the composition and the functionality of lymphocytic infiltration in early-stage TNBC.
Discussion
Recent evidence suggests that the presence of TILs is an important predictor of outcome and response to chemotherapy in TNBC [
3‐
7]. In this dataset, we confirmed that increasing stromal TILs is an independent prognostic marker for prolonged RFS and OS in TNBC treated with adjuvant anthracycline-based chemotherapy. Studies evaluating the association between LPBC and survival have reported conflicting results [
3‐
7]. Indeed, LPBC was not significantly associated with prognosis, likely due to the reduced number of events, and the small proportion of TNBC displaying this phenotype. Thus, further efforts are needed to improve the quantitative pathological assessment of TILs on HE-stained slides.
In agreement with recent data, we demonstrated that a value of ≥20% of stromal TILs could identify a group of low-risk patients with both lymph node-negative and lymph node-positive early-stage TNBC [
17]. Furthermore, our findings suggest that patients with low numbers of TILs may benefit from the generation of an anti-tumor immune response, while boosting the lymphocyte activity (e.g. checkpoint inhibitors) might prove useful in patients with high numbers of TILs, associated with higher disease burden.
Although the presence of TILs reflects the activation of a local anti-tumor immune response, distinct immune cell subpopulations may have specific biological significance. In agreement with previous findings, we demonstrated that both CD8+ and FOXP3+ cells were associated with good outcome in patients with TNBC, and that the clinical significance of FOXP3+ lymphocytes was highly dependent on the concurrent presence of cytotoxic T cells [
23‐
27]. Interestingly, when stratified based on the presence of CD8+ lymphocytes, a high infiltration of FOXP3+ cells trended towards reduced survival in TNBC patients with low numbers of CD8+ cells. These results suggest that CD8+ lymphocytes could be the main effectors of anti-tumor immune responses, and that the consistent correlation between FOXP3 positivity and cytotoxic lymphocytes may in part explain conflicting results reported in previous studies [
8].
Overall, our findings indicate that the assessment of single immune components may not be as informative as the global evaluation of stromal TILs. However, the understanding of the biological role of different lymphocyte subpopulations warrants further investigations, and could be useful in selecting patients with TNBC who may benefit from the addition of specific immunomodulatory therapies to conventional chemotherapeutic regimens.
Even though TILs are emerging as important prognostic and predictive factors in TNBC, it is worth noting that many TNBC have few TILs, and even in the presence of massive lymphocytic infiltration, immunosuppressive mechanisms should be considered [
28]. In this scenario, both radiotherapy and chemotherapeutic agents (e.g. anthracyclines) have been shown in preclinical models to be able to shape the tumor microenvironment, and to boost an effective immune response against tumor cells [
29,
30]. These therapies could be rationally evaluated in combination with immunomodulatory drugs to synergize with pre-existing lymphocytes with tumoricidal activity, or to elicit a
de novo local immune response in tumors lacking TILs.
Even though the immune system can recognize and eliminate malignant cells, tumors have developed multiple mechanisms to evade effective immunosurveillance, including the activation of the immune checkpoints PD-1 and LAG-3 [
2,
9]. We demonstrated that PD-1+ and LAG-3+ TILs were present in approximately 30% and 18% of TNBCs, respectively, and that their presence in the tumor microenvironment tended to be associated with good prognosis in TNBC. The upregulation of these receptors, especially PD-1, has been classically described as a prominent immune resistance mechanism, and analyses performed on tissue microarrays have revealed an inverse correlation with outcome in patients with breast cancer [
9,
31]. Indeed, double-positive PD-1/LAG-3 TILs have been recently demonstrated to show a more exhausted phenotype and functionality compared with single-positive or negative TILs in a preclinical model, likely leading to increased cancer immune evasion [
32]. However, the role of co-inhibitory molecules in the modulation of the tumor immune microenvironment, and the mechanisms underlying T cell exhaustion and anergy are still poorly understood [
9,
33]. Furthermore, it is worth noting that the activity of immune cells depends on the interaction with cancer cells, and recent findings support the idea that the functional relevance of checkpoint proteins is highly sensitive to the context (e.g. amount of antigen, topographical relationships with tumor cells and PD-L1-expressing cells) [
9,
34]. Consequently, the evaluation of the clinical and biological significance of immune markers, especially those reflecting the activation status of lymphocytes, should be performed on whole tissue sections, reducing sampling bias due to tumor heterogeneity, and providing a more comprehensive understanding of the complex tumor-immune dynamics. Moreover, we found that the expression of both PD-1 and LAG-3 highly correlated with the presence of TILs, especially cytotoxic CD8+ cells.
Even though stratified analysis according to levels of lymphocytic infiltration was not performed due to the low number of cases in each subgroup, our results suggest that the presence of PD-1+ and LAG-3+ TILs in the tumor microenvironment may reflect the occurrence of an active, although partially exhausted, intratumoral immune response, rather than representing a global marker of immunosuppression. Accordingly, emerging evidence have revealed that local immunomodulatory factors (e.g. IFN-γ released by TILs), or the activation of oncogenic signaling pathways (e.g. the PI3K pathway) can promote the expression of PD-L1, which has been shown to be enriched in triple-negative/basal-like breast cancer, and to be associated with good outcome and response to chemotherapy in patients with TNBC [
6,
10‐
14].
Interestingly, by analyzing two independent cohorts, we found that PD-1+ and LAG-3+ TILs were concurrently expressed in approximately 15% of TNBC cases. Thus, reversing the phenotype of exhausted T lymphocytes by targeting multiple inhibitory receptors may boost an effective anti-tumor immune response, and represent a novel valuable strategy to treat a subgroup of patients with TNBC. Recently, the blockade of the PD-1/PD-L1 pathway has shown promising clinical activity in patients with metastatic TNBC, although molecular preselection of the candidate patients for novel clinical trials would be valuable [
33,
35‐
38]. Preclinical data demonstrates that anti-LAG-3 is mildly effective as monotherapy, but potently synergizes with anti-PD-1, suggesting that the combined immune checkpoint inhibition could enhance T cell activity and improve anti-tumor immunity [
32]. Furthermore, the dual blockade of PD-1 and LAG-3 may exhibit less immune toxicity than that observed with the blockade of other immune receptors (e.g. CTLA-4).
Even though we confirmed the prognostic value of TILs in TNBC, the biological link between FOXP3+ and CD8+ lymphocytes, and the clinical relevance of checkpoint receptors in patients with TNBC with different levels of TILs, warrant further investigations. Despite these potential limitations, our findings support the clinical evaluation of combination immunotherapies with anti-PD-1/PD-L1 and anti-LAG-3 in a specific subset of patients with TNBC who have concurrent expression of both checkpoints.
Acknowledgments
The authors are grateful to all colleagues working at the Breast Unit at Humanitas Clinical and Research Institute (Rozzano - Milan, Italy), and Humanitas Oncology Center of Catania (Catania, Italy) and Humanitas Mater Domini (Castellanza, Italy).